Development, Characterization, and Standardization of a Nose-Only Inhalation Exposure System for Exposure of Rabbits to Small-Particle Aerosols Containing Francisella tularensis

Abstract

Inhalation of Francisella tularensis causes pneumonic tularemia in humans, a severe disease with a 30 to 60% mortality rate. The reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing F. tularensis We evaluated the relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize the spray factor (SF), a central metric of aerosolization. This optimization reduced both inter- and intraday variability and was applicable to multiple isolates of F. tularensis Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of the bacterial culture optical density and the number of CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Live vaccine strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent F. tularensis SCHU S4 that had been cultivated in either Mueller-Hinton broth (MHB) or brain heart infusion (BHI) broth. LVS-vaccinated animals challenged with SCHU S4 that had been cultivated in MHB experienced short febrile periods (median, 3.2 days), limited weight loss (<5%), and longer median survival times (∼18 days) that were significantly different from those for unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with SCHU S4 that had been cultivated in BHI experienced longer febrile periods (median, 5.5 days) and greater weight loss (>10%) than the unvaccinated controls and median survival times that were not significantly different from those for the unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents.

Keywords: Francisella tularensis; aerosols; animal models; inhalation; rabbits; tularemia.

FIG 1

Aerosol performance of the RNO exposure chamber. (A) Change in relative humidity at 5-s intervals over 10 consecutive 10-min aerosol exposures in the RNO exposure chamber with F. tularensis in BHI. (B) Aerosol particle size in the RNO exposure chamber, as measured by the aerodynamic particle sizer using 1-μm fluorescein microspheres. The solid black line is the relative mass of the particles, as measured by the concentration (left y axis) compared to the particle size (x axis); the dotted line is the cumulative percentage of aerosol particles (right y axis) compared to the particle size (x axis). GSD, geometric square deviation. (C) SF for F. tularensis SCHU S4 in the RWB and RNO exposure chambers after being grown overnight in either BHI or MHB. Asterisks indicate results that were significantly different (**, P < 0.05) or highly significantly different (***, P < 0.001) by one-way ANOVA with multiple comparisons.

FIG 2

Variation between aerosol exposures of F. tularensis SCHU S4. (A) The graph shows the SF for individual F. tularensis aerosol exposures (gray circles) on 14 different days, with the mean and standard deviation (lines and error bars, respectively) being shown for each day. The dotted line shows the mean SF (9.48 × 10⁻⁷) across all SCHU S4 aerosols. (B) The graph shows the SF for aerosol runs over multiple days; the values plotted are the individual SF for that run from different days with the mean and standard deviation. The dotted line shows the mean SF (9.48 × 10⁻⁷) across all SCHU S4 aerosols.

FIG 3

No significant variation in SF between different isolates of F. tularensis. SCHU S4, LVS, or recombinant derivative isolates of SCHU S4 were grown in BHI and aerosolized in the RNO exposure chamber. The results shown are the individual SF for different exposures with the mean and standard deviation for each isolate. The dotted line shows the mean SF (9.48 × 10⁻⁷) for SCHU S4 aerosols.

FIG 4

Improvements in inhaled dose based on improved determination of bacterial concentration using the OD₆₀₀ within the linear range. (A) Aerosol doses in rabbits for the first vaccine study (gray circles are results for individual rabbits); the actual dose is on the y axis, while the target dose is on the x axis. (B) OD (x axis) and CFU (y axis) readings of different cultures of SCHU S4 grown in BHI used to determine the nebulizer concentration for the results shown in panel A. (C) Narrowed OD-to-CFU range to fit linear regression analysis; the resulting formula is shown on the graph. The r² value was 0.99. (D) Aerosol doses in the subsequent vaccine study (gray circles are the results for individual rabbits); the actual dose is on the y axis, while the target dose is on the x axis.

FIG 5

Averaged real-time minute volume in rabbits during aerosol exposures to F. tularensis. (A) Averaged minute volume (y axis) over 10-min aerosol exposures for individual rabbits (gray circles) compared to the baseline weight (x axis); the black line is the minute volume calculated from body weight using Guyton’s formula. (B) Averaged minute volume for individual rabbits on the day of exposure. The dotted line is the mean minute volume across all rabbits.

FIG 6

BHI-grown F. tularensis is a more rigorous challenge than MHB-grown F. tularensis for vaccinated rabbits. Rabbits were vaccinated by aerosol prime-boost delivery of LVS 2 weeks apart and then challenged 30 days after the boost with ∼500 CFU of SCHU S4 grown in MHB (black lines, A and C) or BHI (gray lines, A and C). (A) Mean body temperature by group with error bars for the standard deviation for the first 20 days postchallenge (x axis); (B) mean body temperature by group, including mock-vaccinated groups, as a heat map for the first 20 days postchallenge; (C) mean percent weight change by group with error bars for the standard deviation for the first 20 days postchallenge (x axis); (D) time to death in each vaccine and control group compared to historical mock-vaccinated controls challenged with equivalent doses of BHI-grown SCHU S4. The time to death for the LVS-vaccinated, MHB-grown SCHU S4-challenged rabbits was significantly different from that for the historical controls by one-way ANOVA (P < 0.05). The time to death for the other groups was not significantly different from that for the historical controls.